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In the past, non-geniculate coralline algae in the northwestern Gulf of Mexico have been identified based primarily on comparative morpho-anatomy. Recent studies employing DNA sequencing techniques combined with morpho-anatomical studies using SEM have revealed a wealth of previously undocumented diversity of rhodolith-forming non-geniculate coralline algae in the Corallinales, Hapalidiales and Sporolithales from mesophotic hard bank communities at 45-90 meters depth. Although many advances in the last decade have been made in clarifying species names and describing new species of corallines from offshore Louisiana and Texas, total diversity estimates are still incomplete and many species remain to be described. Collections from offshore Louisiana at Parker Bank in the newly expanded Flower Garden Banks National Marine Sanctuary yielded thin, finely branched rhodoliths. DNA sequence analyses of plastid-encoded psbA and rbcL loci, and nuclear-encoded LSU rDNA of these rhodolith-forming specimens revealed that some belong to an unnamed species of Sporolithon (Sporolithales) that we herein newly describe. Additionally, comparative DNA sequence analyses of rhodolith collections from Ewing Bank and other hard banks offshore Louisiana were conducted to assess rhodolith diversity in these mesophotic communities. The results revealed new reports of taxa for the region, including new rhodolith-forming species of Roseolithon (Hapalidiales) to be described herein as well. Our new biodiversity findings will be compared with historical studies from the NW Gulf of Mexico.

The green macroalga Caulerpa ashmeadii was recently found in the North-eastern Atlantic Ocean, on the southern coast of Porto Santo Island (Madeira archipelago). Caulerpa ashmeadii was detected in a subtidal soft-bottom forming scattered patches, mostly among the green calcareous macroalgae Halimeda incrassata and Penicillus capitatus, within a depth range of 18–26 m. Some isolated patches were also observed in bare sand. The species is currently distributed over an estimated area of approximately 0.22 km2. Caulerpa ashmeadii may represent a recent settler (Nov. 2020), although the hypothesis of the species being present elsewhere in Porto Santo prior to our discovery cannot be discarded. Alternatively, the presence of C. ashmeadii in Porto Santo may indicate the influence of climate change on the composition and structure of benthic communities in the Webbnesia region, which further stresses the importance of conducting more intensive biological and environmental monitoring programmes. Overall, this new record constitutes an exceptional eastern expansion of this marine species by more than 4200 km, comprising its first register in the Eastern Atlantic Ocean.

Additional file 8: Figure S6. Bin reciprocity. Sankey plot depicting MyCC bin correspondence across Illumina and hybrid assembly files (Illumina PE + 1Df or 2D) and their taxonomy/origin in the Caulerpa ashmeadii holobiont based on COGs. Flow size linking bins represent the number of common scaffolds in the assembly files compared via reciprocal BLASTn. Numbers adjacent to bins represent the number of COGs reported by MyCC. Note the consistent delimitation of nuclear contigs but some instability in the binning of the Phyllobacteriaceae sp. and Rhodospirillaceae sp2. scaffolds. The Sankey diagram was built on the basis of html code available from Google Charts at https://developers.google.com/chart/interactive/docs/gallery/sankey. PE = Illumina Paired-Ends scaffolds, PE+1Df = hybrid 1Df scaffolds, PE+2D = hybrid 2D scaffolds.

Additional file 4: Figure S2. Assembly file content. Contig/scaffold abundance per genomic compartment across nanopore and hybrid assemblies. Bubble size represent the percentage of uniquely mapped reads to a given assembly and corresponding dataset (those representing

Additional file 6: Figure S4. Circular Mitochondrion genome map.

Additional file 2. Supplementary tables.

Additional file 9: Figure S7. Metagenome binning. Comparison of delimited bins with MyCC (4mer) for three assemblies. (a) Experimental binning of Canu 1Df contigs. (b) Binning of Spades’ hybrid Illumina+1Df assembly. (c) Binning of Spades’ Illumina-only assembly. Bins containing nuclear and repeat contigs are emphasized with dashed circles. Remaining bins correspond to bacterial taxa. Note that printed bin numbers are unrelated between plots.

Additional file 12: Figure S10. BLASTn report for atp1/atpA. BLASTn alignment of mitochondrial atp1 and chloroplast atpA. Note identity level nearing ~ 70%, causing mismapping of low quality atpA reads on atp1.

Additional file 7: Figure S5. GenomeScope profile. Kmer coverage and log coverage for the entire Illumina dataset (a and b), the dataset depleted of bacterial reads (c and d), and the dataset depleted of nuclear reads (e and f). Note the drop in kmer frequencies indicated by arrows and circles to point at the depletion of bacterial kmers overlapping with nuclear ones (a and c) and their recovery in the dataset depleted of nuclear reads (e). Note as well the successful depletion of the main bacterial kmer peak (*) in the bacterial depleted dataset (a and e vs. c).

Additional file 11: Figure S9. Polished quality of protein-encoding gene. Detailed distributions of gene quality following polishing steps as (a) boxplot or (b) violin plot. For comparison, numbers reported between parenthesis in Table 4 correspond to the cumulative percentage identity, indels and substitutions for all genes, while the data points and distribution in the present figure represent individual gene values. Note the perfect quality of chloroplast genes following Pilon polishing. CP_1DF=Chloroplast genes from 1Df assembly, CP_2D = Chloroplast genes from 2D assembly, MT_1Df = Mitochondrion genes from 1Df assembly.

Additional file 17: Figure S15. BLASTp report for ORF7, ORF8, and ORF9.

Additional file 15: Figure S13. Chloroplast genome long reads’ pile-up.

Additional file 16: Figure S14. BLASTp report for psbA intronic ORFs.

Additional file 10: Figure S8. Phylogeny of valyl-tRNA ligase. Exploratory phylogeny of valyl-tRNA ligase (COG0525) amino acid sequences extracted from nuclear and bacterial bins by MyCC. For taxonomic identification of bins, sequence context was retrieved via BLASTp against GenBank’s non-redundant protein databases. Note the occurrence of two different valyl-tRNA ligases in Caulerpa ashmeadii nuclear genome.

Additional file 5: Figure S3. Circular Chloroplast genome map.

Additional file 14: Figure S12. Error rate vs. assembly. Impact of Canu’s error rate parameter on assembly of the circular chloroplast genome in the presence of structural variation (SV) (black bubbles). Note the occurrence of misassemblies (i.e. polymorphism/SVs stitched on the contig’s extremities) when using relaxed error rates with 2D data, while 1D assembly still produces a circularizable contig (i.e. no misassembly).

Additional file 3: Figure S1. Read length distribution. Density plot depicting read length improvement following decontamination of low molecular weight fragment (LMW). LibRAW: original DNA extract, LibGEL: Gel excised HMW DNA, and LibMAG: HMW DNA selected via 0.4X magnetic bead wash. Data is shown for 1Df and 2D reads. Note the broader shoulder of LibGEL for sequence > 2500 bp.

A second species in the siphonous green algal genus Avrainvillea was recently discovered off the island of O'ahu in the Main Hawaiian Islands. Specimens were collected from Honolulu Harbor, including its entrance channel, and near Ke'ehi Harbor. These locations are both in Mālama Bay on O'ahu's south shore in or adjacent to urbanized estuaries, respectively. In situ observations, morphological and molecular assessments were conducted to examine the alga's habit and distribution, as well as to assess its putative species identification. The alga occurred in sand as single individuals or in clusters of several individuals at both sites, and near or within seagrass beds (Halophila decipiens) and algal meadows composed of the green alga Halimeda kanaloana and an unidentified Udotea species at the Ke'ehi Harbor site. All analyses supported both populations as representative of the same taxa, reported until further investigation in the broad Pacific as Avrainvillea cf. erecta based on morphological and molecular analyses. This record of a second Avrainvillea species in Hawai'i is of particular concern considering that an alga recognized as A. amadelpha, first observed in 1981 from two locales on O'ahu's south shore, has become invasive in Hawai'i's intertidal to mesophotic environments.

Additional file 1. FigTree Tutorial and command line usage for TREE2FASTA.

Additional file 5. Illustration of the edited exploratory tree used to measure TREE2FASTA execution speed reported in Table 1. Both trees were edited for color and/or annotation in FigTree for 10 phyla of the tufA and 16S PhytoREF database. Color details: Chlorarachniophyta (CYAN), Chlorophyta (GREEN), Euglenozoa (BLUE), Cryptophyta (BROWN), Rhodophyta (RED), Glaucophyta (MAGENTA), Ochrophyta (ORANGE), Haptophyta (YELLOW), Cyanophyta (BLACK), Streptophyta (PURPLE). Note that smaller phylum clades, albeit present in both trees are not easily visible among other colors (e.g. Cryptophyta, Euglenozoa). Cyanophyta is absent from the PhytoREF. Streptophyta is absent for the tufA database.

Additional file 2. Details of color and annotation information saved within the TAXA block of FigTreeâ s NEXUS file.

Additional file 4. Details of the Florideophyceae clade shown in Fig. 2a.

Additional file 3. Example command line for multiple alignment in MUSCLE and maximum likelihood estimation RAXML to rapidly produce exploratory tree.

Additional file 12: Figure S5. Example of putative index tag jumping or tag error. Comparative read abundance for seven OTUs demultiplexed (i.e. assigned) to both FL02 and JP07. For illustration purpose, only OTUs with >5 % of mapped reads in either FL02 or JP07 are shown (these correspond to seven out of the 141 common OTUs, see Additional file 9: Figure S2). Note the drastic abundance disparity illustrative of or potential of false assignment via index tag jumping or tag error (e.g. OTU 511 representing ≪0.1 % reads in FL02 and 10.4 % of reads in JP07). By contrast, note the more balanced abundance of OTU 355, 356, and 506, which may reflect the true presence of common OTUs in both samples (i.e. pantropical OTUs).

Additional file 13: Figure S6. OTUs vs. clone identity and coverage. Comparison of all phototrophic clones obtained from JP07 with their matching OTUs. Note high identity of clones and OTUs based on 375Â bp, with 0, 1 or 2 base pair variation (100, 99.7 and 99.5Â % respectively). Also note that clone H and its corresponding OTU_364 share the highest coverage, although further cloning would be necessary to accurately assess congruence in sequence coverage between the two methods.

Additional file 5: Table S5. Clone taxonomy. Detailed reference taxonomy for 86 phototrophic clones from microfloras sampled in the Ryukyu archipelago (JP01, JP03, JP04, JP06, JP07, JP25), an aquarium window scrap (E09) and a Gulf of Mexico crustose coralline Lithophyllum sp. rhodolith. Taxonomic groupings with unsettled naming such as combined orders, provisional families, and polyphyletic or monophyletic species or genera complexes, are noted between quotes.

Additional file 7: Table S6. Summary of microfloras richness and abundance at multiple taxonomic levels. (A–B) OTU counts and read abundance for ‘Phototrophs’ vs. ‘Heterotrophs’ group. (C–D) OTU counts and read abundance for phototrophic phyla and classes. (E–F) OTU counts and read abundance for Ulvophyceaen families. Note that OTUs may be found more than once across samples.

Additional file 3: Table S3. Indexed forward primers used for metabarcoding. Indexes in bold.

Additional file 2: Table S2. Barcoded Florideophyceae, Phaeophyceae and miscellaneous taxa. Collection information for specimens of the Florideophyceae and Phaeophyceae sequenced for tufA in the present study (as well as a miscellaneous Prasinophyceae). Polyphyletic orders or family and combined orders are indicated in between quotes. All specimens from shallow waters (0–10 m) otherwise indicated in footnotes. Collector (Coll.) initials as follows: CP = C. Pueschel, CS = C. Stoude, DG = D. Gabriel, DK = D. Krayesky, DWF = D.W. Freshwater, EC = E. Coppejans, ED = E. Deslandes, JC = J. Cabioch, JH = J. Hughey, JR = J. Richards, JRu = J. Rueness, JZ = J. Zertuche, MG = M. Guiry, MHH = M. H. Hommersand, MJW = M. J. Wynne, MY = M. Yoshizaki, OC = O. Camacho, SF = S. Fredericq.

Additional file 4: Table S4. Summary taxonomy and content of the non-redundant tufA database. Diversity and sequence counts for (A) Heterotrophs and Phototrophs, (B) Phyla and Classes for all phototrophs and (C) Orders/suborders and families for the Ulvophyceae (Genus taxonomy not shown for conciseness). Taxonomic groupings with unsettled naming such as combined orders, provisional families, and polyphyletic families (i.e. ‘Kornamanniaceae’ and ‘Ulothrichaceae’) are noted between quotes.

Additional file 8: Table S7. OTUs taxonomy and their microflora assignment. Detailed reference taxonomy for 802 core OTUs as determined from their phylogenetic position (see Fig. 6). Taxonomic groupings with unsettled naming such as combined orders, provisional families, and polyphyletic or monophyletic species or genera complexes, are noted between quotes.

Morphological and molecular evidence is provided to further document the status of the enigmatic taxon known as Caulerpa floridana W.R. Taylor from White Shoal, Dry Tortugas, Florida. DNA sequencing of three historical herbarium specimens (WRT329, WRT345 and WRT349) housed at the University of Michigan Herbarium (MICH) demonstrated the molecular separation of this species based on the reconstruction of 931 nucleotides of the chloroplast gene tufA. Caulerpa floridana is sister to the western Atlantic endemic C. ashmeadii Harvey and an unknown Caulerpa taxon from the Florida Middle Grounds. Caulerpa floridana most reliably differs from C. ashmeadii by the presence of a sharp, unequivocal apiculus at the tip of each pinnule. A morphological review of southwestern Atlantic records of C. floridana from Brazil excludes these reports as representative of the species.

Additional file 1: Table S1. Barcoded Ulvophyceae. Collection information for Ulvophyceae specimens newly sequenced for tufA. To ease public database searches, Genbank® identifiers include both morphological and molecular identification (in brackets []) at the genus or family level (when abbreviated). Polyphyletic genera, species complexes or combined orders indicated in between quotes. All specimens from shallow waters (

Additional file 1: Table S1. Barcoded Ulvophyceae. Collection information for Ulvophyceae specimens newly sequenced for tufA. To ease public database searches, Genbank® identifiers include both morphological and molecular identification (in brackets []) at the genus or family level (when abbreviated). Polyphyletic genera, species complexes or combined orders indicated in between quotes. All specimens from shallow waters (